1. Volume 17, Issue 8, Pages 1465-1472 (August 2009)
Recombinant Vesicular Stomatitis Virus Transduction of Dendritic Cells Enhances Their Ability to Prime Innate and Adaptive Antitumor Immunity 
Jeanette E Boudreau, Byram W Bridle, Kyle B Stephenson, Kristina M Jenkins, Jérôme Brunellière, Jonathan L Bramson, Brian D Lichty, Yonghong Wan 
Molecular Therapy 
Volume 17, Issue 8, Pages (August 2009)
DOI: /mt
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
DCs are efficiently transduced using VSV and remain viable for at least 72 hours after infection. (a) Triplicate wells of DCs were infected with 0 (i) (mock treatment), 10 (ii), 25 (iii), or 50 (iv) pfu/cell VSV/GFP for 4 hours in minimal media, washed thrice in PBS, and returned to culture in DC media. Representative dot pots depicting GFP expression versus CD11c are shown. (b) GFP expression by DCs (gated on CD11c+ cells) was quantified 24 hours postinfection by flow cytometry (*P < compared to all other groups). (c) Cell viability was determined by 7-amino actinomycin-D staining 24, 48, or 72 hours postinfection or mock treatment. (d) Samples of infected cell supernatants were collected 24, 48, and 72 hours after infection and washing and viral progeny was quantified by plaque assay. Vero cells were infected using identical conditions as a positive control for productive infection. Data are representative of three independent trials. APC, allophycocyanin; DC, dendritic cell; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; PBS, phosphate-buffered saline; pfu, plaque-forming units; VSV, vesicular stomatitis virus.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
VSV infection induces maturation and cytokine production by DCs. DCs were infected with 25 pfu/cell VSV/MT, treated with LPS, or mock-treated for 4 hours in minimal media, washed thrice in PBS, and returned to culture. (a) Representative histograms depicting CD40, CD86, and MHC II expression from mock (shaded) and stimulated (black line) samples are shown. (b) Summarized data for CD40, CD86, and MHC II expression by DCs (**P < compared to mock-treated cells; *P < 0.05, significantly different from mock- and VSV-treated cells). (c) 12 hours following infection, intracellular staining for IL-12 and TNF-α was performed (**P < 0.001, significant increase compared to mock-treated cells). (d) Supernatants were collected 12 hours after infection and analyzed for acid-resistant type-I IFN based on VSV plaque reduction assay as described in materials and methods (*P < 0.05 compared with mock- and LPS-treated cells). Bars represent means ± SEM and data from three independent infections. DC, dendritic cell; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; MHC, major histocompatibility complex; pfu, plaque-forming units; TNF, tumor necrosis factor; VSV, vesicular stomatitis virus.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
VSV-infected DCs mediate therapeutic tumor destruction. (a) Mice were challenged with 1 × 106 B16-OVA cells intravenously and immunized 10 days later with PBS, DC-VSV/MT, or DC-VSV/SIIN. Mice were killed 11 days after immunization (21 days after challenge), lungs were collected and tumour nodules were counted using a dissecting microscope (significantly greater tumor burden in PBS compared to DC-VSV/MT (**P < 0.001) and DC-VSV/SIIN (**P < 0.001) *P < 0.05, significant difference between DC-VSV/MT and DC-VSV/SIIN). (b) Mice were immunized with DCs treated LPS with or without SIINFEKL peptide pulsing, or infected with recombinant Ad carrying SIINFEKL (DC/Ad-SIIN) or no transgene (DC/Ad-BHG). Significant reduction compared to PBS, **P < Bars represent means ± SEM from two independent experiments with 5 mice/group. DC, dendritic cell; LPS, lipopolysaccharide; OVA, ovalbumin; PBS, phosphate-buffered saline; VSV, vesicular stomatitis virus.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Immunization with VSV-infected DCs elicits NK and CTL activation in tumor-bearing hosts. Mice bearing 10-day-old B16-OVA lung metastases were immunized with DC-VSV/SIIN or DC-VSV/MT, or unimmunized (PBS). Eleven days following immunization, splenic NK cells were analyzed for activation by flow cytometry based on (a) CD69 expression and (b) IFNγ production (***P < , compared with PBS-immunized). (c) To analyze CTL activation, IFNγ production following in vitro peptide restimulation was measured by flow cytometry (*P < 0.05 significantly greater IFNγ production following restimulation with SIINFEKL peptide compared to PBS and DC-VSV/MT-immunized mice; **P < 0.001, significantly greater production of IFNγ following restimulation with RGYVYQGL peptide compared to PBS-immunized. (d) In vivo T-cell cytotoxic activity was measured as described in materials and methods (***P < , significantly greater target cell killing compared to unpulsed targets. Bars illustrate means ± SEM. Data are representative of two independent experiments with 5 mice/group. CTL, cytotoxic T-lymphocyte; DC, dendritic cell; IFN, interferon; NK, natural killer; OVA, ovalbumin; PBS, phosphate-buffered saline; VSV, vesicular stomatitis virus.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Tumor destruction mediated by VSV-infected DCs involves both CD8+ and NK cells. Mice were challenged and immunized as described in Figure 3. CD8 and NK-depleting antibodies were administered throughout the immunization period the number of lung tumor metastases was enumerated 21 days after challenge (*P < 0.01 and **P < compared to DC-VSV/SIIN-immunized). Bars represent means ± SEM and two independent experiments with 5 mice/group. DC, dendritic cell; NK, natural killer; VSV, vesicular stomatitis virus.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
DCs infected with VSV carrying a native tumour antigen mediate tumor protection. Mice were challenged with 106 B16-OVA cells i.v., and treated with PBS or immunized with DC-VSV/hDCT. Eleven days after immunization, mice were killed and spleens and lungs were collected. (a) Tumor nodules on lung surfaces were counted using a dissecting microscope (significantly greater tumor burden in PBS-treated compared to DC-VSV/MT (**P < 0.01) and DC-VSV/hDCT (**P < 0.001); *P < 0.05, significantly different tumor burdens between DC-VSV/MT and DC-VSV/hDCT). (b) IFNγ production by splenic NK cells was measured after in vitro culture without stimulation (*P < 0.05 compared to PBS-treated). (c) CD8+ T cells were restimulated with SVYVYQGL peptide in vitro and IFNγ production was measured (*P < 0.05 compared to PBS-treated). DC, dendritic cell; IFN, interferon; OVA, ovalbumin; PBS, phosphate-buffered saline; VSV, vesicular stomatitis virus.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene & Cell Therapy Terms and Conditions